qHTS Assay for Modulators of miRNAs and/orActivators of miR-21

MicroRNAs (miRNAs) are endogenously encoded small (~20-25 nucleotides), nonprotein-coding RNAs that are involved in post-transcriptional repression of target messenger RNAs (mRNAs) (Le and Hannon, 2004). Estimated to be involved in the regulation of 30% of all protein-coding mRNAs (Filipowicz et al., 2008), miRNAs play a significant role in many biological processes including cellular more ..

MicroRNAs (miRNAs) are endogenously encoded small (~20-25 nucleotides), nonprotein-coding RNAs that are involved in post-transcriptional repression of target messenger RNAs (mRNAs) (Le and Hannon, 2004). Estimated to be involved in the regulation of 30% of all protein-coding mRNAs (Filipowicz et al., 2008), miRNAs play a significant role in many biological processes including cellular differentiation and tissue development (Ghildiyal and Zamore, 2009). Not surprisingly, miRNAs have been found to be oncogenic or tumor suppressive, with altered miRNA expression levels associated with human cancers (Esquela-Kerscher and Slack, 2006). The miRNA miR-21 has been found to be expressed in most human tissues (Sonkoly et al., 2007), and its overexpression has been associated with a variety of human tumors (Tong and Nemunanitis, 2008). miR-21 has been shown to be anti-apoptotic: knock-down of miR-21 in a glioblastoma cell line was found to activate caspases and lead to apoptosis (Chan et al., 2005). Identification of small molecules that modulate miR-21 specifically, or miRNAs generally, would prove useful as tools to better understand miRNA biogenesis and mRNA regulation by miRNAs. Of special interest are compounds that specifically inhibit miR-21, and a cell-based firefly luciferase reporter gene assay optimized for 1536-well format quantitative HTS (qHTS) has been developed in an effort to identify such compounds.

NCGC Assay Protocol Summary:Reagents: HeLa cells stably expressing firefly luciferase (FLuc) under the control of a CMV promoter (upstream) and a miR-21 binding sequence directly downstream of the gene. These cells endogenously express miR-21. Control compounds used were compound C3 (provided by collaborator), N6-phenyladenosine (MP Biomedicals, 0215379625), and DMSO.Assay Summary: 6uL of HeLa miR-21-FLuc cells in assay buffer (DMEM, Invitrogen #21063-029; 10% FBS, HyClone #SH30071.03) were dispensed at 2000cells/well (3.3 x 105 cells/mL) into Greiner solid-white TC-treated 1536-well plates using a Multidrop Combi (Thermo Fisher Scientific). Plates were incubated for one hour at 37 degree Celsius, 95% humidity, 5% CO2. These assay plates were then treated with 23nL of compound or DMSO using a Kalypsys pin tool, which allows for delivery of a 24-point titration of each compound to the assay plate, with a final compound concentration ranging from approximately 40uM to 5pM. Assay plates were then incubated for 48 hours. After incubation, plates were allowed to come to RT for one hour prior to delivery of 3uL of 3X luciferase detection reagent. After a 15 minute incubation to allow for cell lysis, luciferase activity was measured using a ViewLux CCD imager (PerkinElmer), with an average exposure time of 2-12 seconds. Keywords: NIH Roadmap, MLPCN, MLSMR, qHTS, miR-21, microRNA, firefly luciferase, FLuc.

Compound Ranking:1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.

Float

μM

3

Efficacy

Maximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.

Float

%

4

Analysis Comment

Annotation/notes on a particular compound's data or its analysis.

String

5

Curve_Description

A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.

String

6

Fit_LogAC50

The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).

Efficacy at zero concentration of compound from a fit of the data to the Hill equation.

Float

%

11

Fit_CurveClass

Numerical encoding of curve description for the fitted Hill equation.

Float

12

Excluded_Points

Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.